Project Summary Human Immunodeficiency Virus-1 (HIV-1) has killed over 35 million people to date and infects 2 million new people each year. Infection with HIV causes depletion of CD4+ T Cells leading to an immunocompromised state, and high plasma viral load is correlated with high transmissibility. Antiretroviral therapy (ART), although effective in controlling plasma viremia and transmission, does not purge the latent or persistent reservoirs necessary to eliminate infection. Given that non-compliance, socio-economic barriers, and loss to follow up are common barriers to successful ART therapy, it is imperative to discover therapeutics that provide both lifetime suppression of viral loads and depletion of viral reservoirs. Recently, studies have demonstrated control of viral replication and decreasing viral reservoirs in 50% of rhesus vaccinated with a CMV vaccine vector. They propose that the continuous immunosurveillance of SIV by TEM cells is maintained by the persistent CMV vectors. To mimic the immunosurveillance and increase the HIV-specific CTL activity in vivo, we will genetically modify CMV-specific T cells with a chimeric antigen receptor (CAR) and follow the effects on viral reservoirs in humanized mice and rhesus macaque. The CARs express the CD4 extracellular domain to redirect CTL activity against HIV, and intracellular T cell signaling domains to stimulate CTL functions. These genetically modified T cells target a critical step in the viral life cycle independent of MHC presentation, targeting heterogeneous viruses while avoiding the potential for viral escape. Additionally, based our preliminary studies, we will include the potent fusion inhibitor maC46 to protect these genetically modified T cells from infection. In contrast to prior studies, maC46 will be incorporated bicistronically, as opposed to co-transduced. This will have the added benefit of higher percentages of transduced cells carrying both CD4-CAR and maC46. Such an innovation would result in greater ability to generate genetically modified T cells when adapted to a clinical setting. We hypothesize that CMV-specific T cells, when transduced with bicistronic CD4-CAR and fusion inhibitor, will persist in vivo based on their CMV specificity, but will be protected from infection and will target residual/reactivated HIV+ cells. These experiments would be the first use of a bicistronic vector incorporating CD4-CAR with maC46 transduced into CMV-specific T cells, and show rescue of both a humanized mouse, and rhesus macaque model. We will address this hypothesis through the following specific aims: 1) Transduce CMV-specific T cells with bicistronic CD4-CAR/maC46 and assess inhibition of viral replication, 2) Adoptively transfer CMV-specific CD4-CAR/maC46 transduced T cells in vivo using NSG mice and challenge with HIV infection, and 3) adoptively transfer CMV-specific CD4-CAR/maC46 transduced T cells into SHIV infected rhesus macaque and compare to the clinical efficacy of ART therapy in terms of plasma viral load, transmissibility, and latent reservoirs. Since rhesus macaque is an important animal model for both HIV pathogenesis and gene therapy, the evaluation of genetically modified T cells in rhesus/SHIV model will translate quickly into the clinic. The ability of genetically modified T cells to control viremia in the absence of ART, especially in the rhesus/challenge model, would be a significant advancement in HIV treatment and would strongly promote a new clinical trial for genetically modified T cells in HIV/AIDS.